The present invention relates to methods and compositions for the treatment of gastrointestinal disorders, cancer, cardiovascular disorders, obesity, benign prostatic hyperplacia, disorders of the lung, disorders of the eye, inflammatory disorders, and other disorders. In particular the invention is useful for the treatment of disorders of the gastrointestinal tract, including constipation, irritable bowel syndrome, inflammatory bowel disease, Crohn's disease, diarrhea, ulcerative colitis and other gastrointestinal digestive or motility disorders. The compounds disclosed herein are peptides and peptide analogues which bind to the cellular receptor protein guanylyl cyclase (GC) or Guanylate Cyclase C (also named GCC, GC-C, Guanylyl cyclase C, GUC2C, GUCY2C, guanylate cyclase 2C, heat-stable enterotoxin receptor, hSTAR, intestinal guanylate cyclase, STAR, STA receptor, guanylate cyclase C receptor, GCCR). In some embodiments, the peptides and peptide analogues are agonists and activate the signaling pathway that is activated by the binding of the natural GCC ligands to GCC. In some embodiments, the peptides and peptide analogues block binding of natural ligands of GCC but do not activate the signaling pathway activated by the binding of the natural GCC ligands to GCC. The compounds may be used either alone or in combination with other compounds.
Guanylate Cyclase C (GCC) is a type 1 (membrane bound) guanylate cyclase. Guanylate Cyclase C receptors (GCCR) are found in a number of different tissues in the human body (Vaandrager, 2002), but it is predominately present in the gastrointestinal tract. Agonists to the human GCCR include the natural peptide hormones Guanylin and Uroguanylin, as well as a number of bacterial peptides, including the ST peptides that are produced by Escherichia coli and other bacteria (Currie et al., 1992; Tian et al., 2008; Giannella & Mann, 2003; Hamra et al., 1993; Forte, 1999; Schulz et al., 1990; Guba et al., 1996; Joo et al., 1998).
GCC regulates the fluid balance, inflammatory processes and the balance of proliferation and differentiation of the epithelium in the intestine (Evan & Vousden, 2001; Eastwood, 1992; Li et al., 2007a; Bharucha & Waldman, 2010; Sharma et al., 2010; Weiglmeier et al., 2010). The intestinal epithelium is dynamic, with a well-defined vertical axis extending from the crypt depths, in the wall of the intestine, to the tips of villi which project out into the lumen of the intestine. Epithelial cells are “born” at or near the bottom of crypts as daughter cells produced by intestinal stem cells. Recent work with lineage tracing in transgenic animals has offered evidence that—at least in the mouse intestine—cells with stem cell characteristics reside in a narrow band a few cell layers above the crypt bottom (Barker et al., 2007). These daughter cells continue to divide (proliferate), and their progeny migrates up the wall of the crypt toward the tip of the villus. Along this migration, the cells shift from proliferation to differentiation to become fully-functional mature enterocytes with the capacity to perform the normal functions of the gut including digestion, absorption and secretion. Once at the tip, these cells slough off into the lumen of the intestine and die. Thus, the intestinal epithelium turns over every three to five days. GCC and its endogenous ligands appear to be one of the factors that mirror the shift of epithelial cells from proliferation to differentiation along the crypt-villus axis. Indeed, GCC ligands inhibit the proliferation of these cells and change their gene expression pattern to a more terminally-differentiated state (Pitari et al., 2001).
The binding of endogenous (uroguanylin and guanylin) and exogenous ligands (the methanol-soluble, heat stable enterotoxins) to the extracellular domain of GCCR activates the intracellular guanylyl cyclase domain of this receptor, producing cGMP. One of the results of this increase in intracellular cGMP is activation of cGMP-dependent protein kinase (CGKII) and subsequent phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR). This phosphorylation of CFTR opens its ion channel with subsequent efflux of chloride ions from the enterocytes, followed by the passage of counterions (i.e. Na) and water into the intestinal lumen. In addition to CFTR, other transporters of electrolytes may also possibly be involved in this process, as well as other receptors (Seidler et al., 1997; Vaandrager et al., 1997).
One of the clinical manifestations of reduced CFTR activity in cystic fibrosis patients is the inflammation of airway passages. This effect may be due to CTFR regulating the expression of NF-kB, chemokines and cytokines. Recent reports have also suggested that the CFTR channel is involved in the transport and maintenance of reduced glutathione, an antioxidant that plays an important role in protecting against inflammation caused by oxidative stress (Colin-Bisello et al., 2005). Enhancement of intracellular levels of cGMP by way of guanylate cyclase C activation would be expected to down-regulate these inflammatory stimuli. Thus, GCC agonists should be useful in the prevention and treatment of inflammatory diseases of the lung (e.g., asthma), bowel (e.g., ulcerative colitis and Crohn's disease), pancreas and other organs.
Guanylin and Uroguanylin mediated signaling via cGMP is important to the normal function of the gut. Guanylin and Uroguanylin serve as paracrine regulators of GCCR activity in the intestine and therefore regulate electrolyte and fluid transport in the GI tract. Abnormalities or disturbance of this process contribute to gastrointestinal disorders such as Chronic Idiopathic Constipation (CIC), Irritable Bowel Syndrome (IBS) and Celiac disease (Collins, 2007; Ramamoorthy et al., 2007; Collins & Bercik, 2009). These receptors also influence inflammatory conditions and cell proliferation, and abnormalities in the process can also lead to conditions such as Inflammatory Bowel Disorders (IBD) or Cancers (Shailubhai et al., 2000; Shailubhai, 2002; Li et al., 2007b; Askling et al., 2001).
Chronic Idiopathic Constipation and Irritable Bowel Syndrome are disorders of the gut that are a cause of discomfort and pain. In these conditions there is no serious inflammatory involvement, although there may be a low grade of inflammation present. The pathology involves altered motility, decreased stool hydration, and visceral sensitivity. Underlying causes may include the involvement of 5-HT (5-hydroxytryptamine, serotonin), which is regulated by cGMP. An alteration in the renewal of the mucosa may also be involved along with a change in the apoptosis rate of cells in the intestinal tissue, which may also influence oncogenic processes (Carrithers, 2003; Bharucha, 2010; Lin et al, 2010). The definition and diagnosis of CIC and IBS have been established in the Rome Criteria (Drossman, 1999). CIC and IBS are classified as a functional gastrointestinal disorders, resulting from a combination of altered bowel motility and an increased visceral sensitivity. In CIC, the bowel motility is lowered and stool hydration is reduced. There are three main subgroups of IBS; constipation dominant, diarrhea dominant, or mixed which alternates between constipation and diarrhea. In all IBS conditions bowel motility is altered and there is an increased visceral sensitivity. Both CIC and IBS are very prevalent condition, affecting at least 10 million people in the United States alone.
Inflammatory Bowel Disease describes a group of disorders where the intestine is inflamed. These include Ulcerative Colitis and Crohn's disease. Ulcerative Colitis is an inflammatory disorder of the colon, although it can also appear in other sections of the intestine. Ulcerative Colitis affects only the mucosa of the intestine. Crohn's disease is a serious condition that affects mainly the colon and ileum, but it can also be found in other parts of the intestine. In Crohn's disease, all layers of the intestine are affected. Depending on the location in the intestine, Crohn's disease can also be called enteritis or colitis.
Diarrheal diseases are the fourth leading cause of mortality worldwide, responsible for about 20 million deaths each year. Such diseases are the leading cause of pediatric mortality worldwide, particularly affecting children under 5 years of age. Further, diarrheal diseases are responsible for a large part of the more prevalent growth retardation observed in children raised in developing compared to developed nations. One major cause of diarrheal disease are organisms producing heat-stable enterotoxins (STs), a family of structurally-related peptides produced by a variety of species including, but not limited to enteric bacteria such as E. coli, Yersinia, Enterobacter, and Vibrio. This family of structurally-related ST peptides is homologous to the endogenous peptides guanylin and uroguanylin produced in the mammalian intestine. ST-producing organisms are a major cause of endemic diarrhea in under-developed countries, the leading cause of travelers' diarrhea, and the leading cause of diarrheal disease in agriculturally-important animal populations (scours) in developed and under-developed countries. It is estimated that the annual incidence of ST-induced diarrheal disease numbers in the billions in animals and humans. ST induces diarrhea by binding to GCC, which is selectively expressed in the brush border membranes of intestinal epithelial cells and is the presumed receptor for the endogenous ligands guanylin and uroguanylin. Interaction of ST, or the endogenous ligands guanylin and uroguanylin, with GCC activates that receptor, resulting in the production of intracellular cyclic GMP. Cyclic GMP, through a signaling cascade, induces the secretion of salt and water into the lumen of the intestine, resulting in diarrhea. It has been suggested that one function for the endogenous ligands guanylin and uroguanylin in normal physiology is the regulation of fluid and electrolyte homeostasis in the intestine, and the hydration of intestinal contents (e.g. stool). Thus, it is possible to use analogues of ST peptides as therapeutic agents to affect the state of, or prevent, many diseases where GCC plays a role.
Overall, it may be concluded that agonists of guanylate cyclase C have potential therapeutic value in the treatment of a number of conditions, including constipation, irritable bowel syndrome, and a wide variety of inflammatory conditions, as well as potential use as anti-metastatic agents in the treatment of cancer. The development of new agonists is therefore of substantial clinical importance.
Natural GCCR Agonist Peptide Species
There are a number of different peptides with similarity to Guanylin and Uroguanylin that have been identified in different animal species, including obvious species orthologs as well as more distant homologs, but they all have significant structure and significant sequence homologies (Schulz, 1992; Krause et al., 1997; Nakazato, 2001). All mammalian Guanylins and Uroguanylins are structurally related peptides, typically 15 to 16 amino acids in length, that contain two disulphide bonds (Forte, 1999; Magert, 1998).
The amino acid sequences for the mature forms of Guanylin (Table 1A), Uroguanylin (Table 1B) in a number of vertebrate species, and of some bacterial ST peptides (Table 1C) are listed in the tables below:
TABLE 1AOverview of Guanylin amino acid sequencesPosition from N-terminus(matureSEQ 1234567891011121314 1516peptide)SpeciesGenbankID NOAmino acid sequence shown in publicationPublicationHumanNP_291031.21PGTCEICAYAACTGCSchulz, 1992ChimpanzeeNW_001230449.12PGTCEICAYAACTGCMacaqueXP_001085421.13PSTCEICAYAACTGCRatCAA47901.14PNTCEICAYAACTGCMouseNP_032216.15PNTCEICAYAACTGCPigNP_001153746.16PSTCEICAYAACAGCCowNP_001192919.17PSTCEICAYAACAGCSheepEF654536.18PSTCEICAYAACAGCDogNP_001185717.19PRSCEICAFAACAGCHorseXP_001503217.110PRMCEIC AFAACAGCG. PandaEFB17789.111PSVCEICAFAACAGCOpossumXP_001381608.1 12SHTCEICAFAACAGCPlatypusXP_001505889.1 13DDLCELCAFAACTGCYNote:Guanylin species contains disulphide bonds between cysteines in position 4 and 12, and between position 7 and 15.
TABLE 1BOverview of Uroguanylin amino acid sequencesPosition from N-terminusSEQ12345678910111213141516SpeciesGenbankID NOAmino acid sequence shown in publicationPublicationHuman14NDDCELCVNVACTGCLMarx et al.,1998ChimpanzeeXP_524686.215NDDCELCVNVACTGCLMacaqueXP_001087987.116NDDCELCVNVACTGCLHorseXP_001497636.117NDDCELCVNVACTGCLCowNP_001192745.118NDDCEL CVNVACTGCSPigNP_001153747.119GDDCELCVNVACTGCSGuinea p.NP_001166429.120NDECELCVNIACTGCRatNP_071620.121TDECELCINVACTGCMouseCAM14649.122TDECELCINVACTGCSheepABR67874.123DDDCELCVNVACTGCHopping m.AAL77417.124TDECELCINVACTGCOpossumAAB00553.125QEDCELCINVACTGCVirginiaOpossumXP_001367002.126QDDCEICINVACTGCshort tailedPlatypusXP_001505889.127NDDCELCTNAACTGCYNote:Mature Uroguanylin contains disulphide bonds between position 4 and 12, and between position 7 and 15.
TABLE 1COverview of ST peptide amino acid sequencesPosition from N-terminusSEQ−112345678910111213141516171819SpeciesID NO:Amino acid sequence shown in publicationPublicationE. coli STa28NTFYCCELCCNPACAGCYE. coli STp29NTFYCCELCCNPACTGCYTakao et al.,1983E. coli STh30NSSNYCCELCCNPACTGCYNair & Takeda,1998V. cholerae31NTIDCCEICCNPFCTGCLNArita et al.,n011991aVibrio32NTIDCCEICCNPFCTGCLNArita et al.,mimicus1991bV. cholerae33NLIDCCEICCNPFCTGCLNTakao et al.,n01(H)1985aV. Cholerae34GNLIDCCEICCNPFCTGCLNYoshino et01al., 1993Y.35VSSDWDCDVCCNPACTGCTakao et al.,enterocolitica1984STaY.36EENDDWCEVCCNPACTGCTakao et al.,enterocolitica1985bSTbY.37GENWDWCELCCNPACTGCDelor et al.,enterocolitica1990STcCtrobacter38NNTTYCELCCNPACTGCGiannella,freundii1995Table with overview of selected known ST peptide species.Mature ST peptides have disulphide bonds between positions 5 and 10, 6 and 14, and 9 and 17.The bacterial ST peptides are structurally different from the Guanylin and Uroguanylin peptides. These peptides are typically from 18 to 22 peptides in length, and contain three disulphide bonds (Ikemura 1984; Nair, 1998). A common core motif of all these bacterial peptides is:
N-tail-Cys-Cys-Xaa-Xaa-Cys-Cys-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Cys-C-tail Where N-tail is the N-terminal tail of the peptide, typically four to six amino acids long, and C-tail is the C-terminal tail of the peptide, typically one amino acid. Xaa can be several different amino acids. While there is some variation in the composition of the Xaa amino acids in these peptides, there is significant sequence homology between them, and the pattern of Cys-Cys-(2 amino acids)-Cyc-Cys-(3 amino acids)-Cys-(2 amino acids)-Cys is quite constant. Bacterial ST peptides are more potent stimulators of the GCCR than are Guanylin or Uroguanylin (Hamra et al., 1993; Fan et al., 1997; Hamra et al., 1997; Santos-Neto et al., 1999; Forte et al., 2000; Pitari et al., 2001). There are a number of different variants of ST peptides produced by various bacteria (Yoshimura et al., 1985). The core active sequence, i.e. the core pharmacophore, of the peptide are the 13 amino acids between the cysteine residues, i.e. the sequence Cys-Cys-Xaa-Xaa-Cys-Cys-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Cys. The activity of the ST peptide is fully retained if this structure is intact. If any of the disulphide bonds is disrupted, the activity of the peptide will be significantly degraded (Yamasaki et al., 1988; Yamasaki et al, 1988, Bull. Chem. Soc. Jpn, 61: 1701-1706), although with at least 2 of the disulphide bonds intact, the peptide can retain a portion of its activity (Tian et al., 2008) (Tian et al, 2008, Biopolymers (Pept Sci) 90: 713-723).
The ST peptide has been analyzed, and analogues have been described in a number of publications (see for instance Currie et al., 2006—WO/2006/086653; Waldman & August, 2006—U.S. Pat. No. 7,097,839; Shailubhai & Jacob, 2010—US 2010/0093635 A1). The published peptides analogues that have been made and tested involve modifications to the peptide in one of four modes: 1) Modifications using natural L-amino acids, 2) Modifications using D-amino acids, 3) Modifications to the cysteine bonds of the peptide, and 4) modifications involving conjugation of polymers to the peptide. None of these modifications have resulted in a peptide with improved properties compared to that of the basic 13 amino acid core pharmacophore, such as improved potency, stability or solubility (Tian et al., 2008).